Human tubby homolog

ABSTRACT

The present invention provides a novel human tubby homolog (NHT) and polynucleotides which identify and encode NHT. The invention also provides expression vectors, host cells, agonists, antibodies, or antagonists. The invention also provides methods for treating disorders associated with appetite and eating.

[0001] This application is a divisional application of U.S. applicationSer. No. 08/812,824, filed Mar. 6, 1997, the contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel human tubby homolog and to the use of these sequences in thediagnosis, prevention, and treatment of appetite and eating disorders,especially anorexia, cachexia and obesity.

BACKGROUND OF THE INVENTION

[0003] Appetite and eating are ultimately under the control of thebrain, specifically the hypothalamus and cerebral cortex; however,psychological, social, and genetic factors also affect this area ofhuman behaviour. The cerebral cortex receives positive signals from thefeeding center which is located in the ventrolateral nucleus of thehypothalamus. The feeding center stimulates eating until it is inhibitedby the satiety center which is located in the ventromedial hypothalamus.Chemically, increased plasma glucose, insulin, catecholamines, andbeta-adrenergic stimulation are known to inhibit the eating behaviour.

[0004] Obesity results from the disregulation of one or more of thecontrols or factors which influence eating. Generally, obesity isdefined as an excess of adipose tissue; and clinically, it is defined asthat amount of adiposity that imparts a health risk. Even mild obesity,at 20% over desirable weight according to standard height-weight charts,may increase the risk for disease and premature death.

[0005] Obesity has a major impact on human health and the US healthcaresystem through its effects on cardiovascular disease, diabetes mellitus,and hypertension. Although less well known, gall bladder problems;hyperlipidaemia; Alstrom, Bardet-Biedl, Cushing's, Froehlich's, andPrader-Willi syndromes; and cancers, such as craniopharyngioma andhypothalamic, pituitary, and islet cell tumors, also fall into thiscategory. Recent studies indicate that lipophilic mutagens stored infatty tissues such as mammary gland adipose cells may serve as theprimary source for p53 mutations that result in cancers of thosetissues.

[0006] The mouse obesity gene, tubby (tub), first reported as anautosomal recessive mutation has been cloned (Coleman, D. L. and E. M.Eicher (1990) J. Hered. 81:421-427; Noben-Trauth K. et al. (1996) Nature534-38; and Kleyn, P. W. et al. (1996) Cell 85:281-90). Althoughdifferent length splice variants are reported for the tub molecule, themutant gene is abundantly expressed in hypothalamus and has a G→Ttransversion which affects the 44 amino acids at the carboxyterminus.The hydrophilicity of the tub protein (pI=9.2) and the absence of signalor transmembrane motifs suggest cytosolic localization. The tub proteinhas two sets of serine residues separated by acidic residues which mayfunction as a hinge, and two potential glycosylation sites, N₂₀₅ andN₄₂₆.

[0007] The tub mutation has been associated with maturity onsetdiabetes, insulin resistance, progressive retinal degeneration andhearing loss. Although zinc binding site motifs are not present, tub hassome homology to mouse phosphodiesterase, and Noben-Trauth et al.suggest that sensory deficits may be associated with cGMP induced,phosphodiesterase mediated apoptotic activity.

[0008] Because of the numerous correlations between obesity, health, andhealthcare, polypeptides related to tubby and the polynucleotidesencoding them satisfy a need in the art by providing compositions usefulin the diagnosis, prevention, and treatment of appetite and eatingdisorders, especially anorexia, cachexia, diabetes, and obesity.

SUMMARY OF THE INVENTION

[0009] The present invention features a novel human tubby homolog,hereinafter designated NHT, and characterized as having similarity tothe mouse tub gene (GI 1279766, SEQ ID NO: 3) and the human tub homolog(GI 1305497, SEQ ID NO: 4).

[0010] Accordingly, the invention features a substantially purified NHThaving the amino acid sequence shown in SEQ ID NO: 1.

[0011] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode NHT. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO: 2.

[0012] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO: 2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO: 2.

[0013] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode NHT. Thepresent invention also features antibodies which bind specifically toNHT, and pharmaceutical compositions comprising substantially purifiedNHT. The invention also features agonists and antagonists of NHT andmethods for using the protein, agonists and antagonists in the treatmentof appetite and eating disorders.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ IDNO: 1) and nucleic acid sequence (SEQ ID NO: 2) of NHT. The alignmentwas produced using MACDNASIS PRO software (Hitachi Software EngineeringCo., Ltd., San Bruno, Calif.).

[0015]FIGS. 2A and 2B show the amino acid sequence alignments among NHT(SEQ ID NO: 1), mouse tub gene (GI 1279766, SEQ ID NO: 3) and human tubhomolog (GI 1305497, SEQ ID NO: 4). The alignment was produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc.,Madison Wis.).

[0016]FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PROsoftware) for NHT (SEQ ID NO: 1) and mouse tub gene (SEQ ID NO: 3),respectively. The positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

[0017] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0018] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0019] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0020] Definitions

[0021] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0022] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0023] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0024] NHT, as used herein, refers to the amino acid sequences ofsubstantially purified NHT obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0025] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR kit (Applied Biosystems, Foster City, Calif.) inthe 5′ and/or the 3′ direction and resequenced, or which has beenassembled from the overlapping sequences of more than one Incyte cloneusing the GELVIEW™ Fragment Assembly system (GCG, Madison, Wis.), orwhich has been both extended and assembled.

[0026] A “variant” of NHT, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0027] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0028] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0029] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0030] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic NHT, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0031] The term “agonist”, as used herein, refers to a molecule which,when bound to NHT, causes a change in NHT which modulates the activityof NHT. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to NHT.

[0032] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to NHT, blocks or modulates the biological orimmunological activity of NHT. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to NHT.

[0033] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of NHT. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of NHT.

[0034] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of NHTor portions thereof and, as such, is able to effect some or all of theactions of chemically or structurally related molecules.

[0035] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding NHT or the encoded NHT.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0036] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0037] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0038] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0039] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0040] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0041] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0042] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0043] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0044] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0045] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ ID NO:1” encompasses the full-length human NHT and fragments thereof.

[0046] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0047] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0048] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0049] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding NHT orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0050] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO: 2 by northern analysis is indicativeof the presence of mRNA encoding NHT in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0051] “Alterations” in the polynucleotide of SEQ ID NO: 2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding NHT including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes NHT (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO: 2),the inability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding NHT (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0052] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind NHTpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0053] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0054] The Invention

[0055] The invention is based on the discovery of a novel human tubbyhomolog, (NHT), the polynucleotides encoding NHT, and the use of thesecompositions for the diagnosis, prevention, or treatment appetite andeating disorders, especially anorexia, cachexia, diabetes, and obesity.

[0056] Nucleic acid sequence encoding the human NHT of the presentinvention were first identified in Incyte Clone 492199 from the HNT2neuronal cell line cDNA library HNT2NOT01) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO: 2, was derived from the following overlapping nucleic acidsequences: Incyte Clones 492199 (HNT2NOT01) and 539855 (LNODNOT02). Inone embodiment, the invention encompasses a polypeptide comprising theamino acid sequence of SEQ ID NO: 1, as shown in FIGS. 1A, 1B, 1C, and1D. NHT is 491 amino acids in length and as shown in FIGS. 2A and 2B,has two potential N-glycosylation sites at N₁₆₂ and N₃₅₂. NHT also haspotential cAMP or cGMP phosphorylation sites at R₂₄₁ and R₃₁₉. NHT haschemical and structural homology with the mouse and human tub genes (SEQID NO: 3 and SEQ ID NO: 4, respectively). In particular, NHT sharesabout 49% identity with the mouse and human tub proteins. NHT and themouse tub protein show sequence related differences in theirhydrophobicity plots (FIGS. 3A and 3B), and the isoelectric point of NHTis 8.4, slightly more neutral than that reported for mouse. NHT wasexpressed in five cDNA libraries; four from brain or neuronal cell lines(CORPNOT02, HNT2RAT02, HNT2NOT01, and BRAITUT22) and one from lymph node(LNODNOT02).

[0057] The invention also encompasses NHT variants. A preferred NHTvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the NHT amino acid sequence (SEQ ID NO: 1). Amost preferred NHT variant is one having at least 95% amino acidsequence similarity to SEQ ID NO: 1.

[0058] The invention also encompasses polynucleotides which encode NHT.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NHT can be used to generate recombinant molecules whichexpress NHT. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO: 2 asshown in FIGS. 1A, 1B, 1C, and 1D.

[0059] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding NHT, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring NHT, and all such variations are to beconsidered as being specifically disclosed.

[0060] Although nucleotide sequences which encode NHT and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NHT under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NHT or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NHT and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0061] The invention also encompasses production of DNA sequences, orportions thereof, which encode NHT and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding NHT or any portion thereof.

[0062] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO: 2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0063] Altered nucleic acid sequences encoding NHT which are encompassedby the invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent NHT. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentNHT. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of NHT is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

[0064] Also included within the scope of the present invention arealleles of the genes encoding NHT. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0065] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE DNA Polymerase (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of recombinantpolymerases and proofreading exonucleases such as the ELONGASEamplification system (GIBCO/BRL, Gaithersburg, Md.). Preferably, theprocess is automated with machines such as the Hamilton MICROLAB 2200(Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200; MJ Research,Watertown, Mass.) and the ABI 377 DNA sequencers (Applied Biosystems).

[0066] The nucleic acid sequences encoding NHT may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0067] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0068] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0069] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0070] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0071] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Applied Biosystems) and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

[0072] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode NHT, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of NHT in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressNHT.

[0073] As will be understood by those of skill in the art, it may beadvantageous to produce NHT-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0074] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterNHT encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0075] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding NHT may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NHT activity, it may be useful toencode a chimeric NHT protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the NHT encoding sequence and theheterologous protein sequence, so that NHT may be cleaved and purifiedaway from the heterologous moiety.

[0076] In another embodiment, sequences encoding NHT may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of NHT, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Applied Biosystems).

[0077] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of NHT, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0078] In order to express a biologically active NHT, the nucleotidesequences encoding NHT or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0079] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding NHTand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0080] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding NHT. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or PBR322 plasmids); or animal cell systems.

[0081] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NHT,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0082] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for NHT. For example, whenlarge quantities of NHT are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asPBLUESCRIPT (Stratagene), in which the sequence encoding NHT may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of 6-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0083] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0084] In cases where plant expression vectors are used, the expressionof sequences encoding NHT may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

[0085] An insect system may also be used to express NHT. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding NHTmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of NHT will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which NHT may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0086] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding NHT may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing NHT in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0087] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding NHT. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding NHT, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0088] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0089] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress NHT may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0090] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0091] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding NHT isinserted within a marker gene sequence, recombinant cells containingsequences encoding NHT can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NHT under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0092] Alternatively, host cells which contain the nucleic acid sequenceencoding NHT and express NHT may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0093] The presence of polynucleotide sequences encoding NHT can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding NHT. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NHT to detect transformantscontaining DNA or RNA encoding NHT. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

[0094] A variety of protocols for detecting and measuring the expressionof NHT, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NHT is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0095] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding NHTinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding NHT, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0096] Host cells transformed with nucleotide sequences encoding NHT maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNHT may be designed to contain signal sequences which direct secretionof NHT through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding NHT tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and NHT may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingNHT and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992) Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying NHT from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

[0097] In addition to recombinant production, fragments of NHT may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Applied Biosystems). Various fragments of NHT maybe chemically synthesized separately and combined using chemical methodsto produce the full length molecule.

[0098] Therapeutics

[0099] NHT shares 49% chemical and structural homology with mouse andhuman tub genes and is expressed in four different brain libraries andin one lymph node library. Therefore, expression of NHT appears to beassociated with mammalian appetite and eating disorders.

[0100] In one embodiment, NHT or a fragment or derivative thereof may beadministered to a subject to inhibit appetite or overeating, and inparticular to treat or prevent a disorder which is associated withovereating. Such disorders may include cardiovascular diseases such asarteriosclerosis, atheroschlerosis, and hypertension, autoimmune orgenetic disorders such as asthma, fatty liver or pancreas, hypoglycemia,hyperglycemia, and diabetes mellitus, gall bladder disease;hyperlipidaemia; Alstrom, Bardet-Biedl, Cushing's, Froehlich's, andPrader-Willi syndromes; sleep apnea; and adenocarcinomas, leukemias,lymphomas, melanomas, or sarcomas, particularly cancers such ascraniopharyngioma, hypothalmic, pituitary, and islet cell tumors, andadenocarcinomas of the brain, breast, gall bladder, liver, pancreas, andprostate.

[0101] In another embodiment, a vector capable of expressing NHT, or afragment or a derivative thereof, may be administered to a subject totreat or prevent a disorder associated with overeating including, butnot limited to, those listed above.

[0102] In another embodiment, agonists which are specific for NHT may beused to stimulate or prolong the activity of NHT and may be administeredto a subject to treat or prevent a disorder associated with overeatingincluding, but not limited to, those listed above.

[0103] In another embodiment, antagonists or inhibitors of NHT may beadministered to stimulate appetite or eating, and in particular, totreat or prevent an eating disorder. Such a disorder may include, but isnot limited to, amenorrhea, anorexia nervosa, bulimia nervosa, cachexia,and loss of appetite associated with chemotherapy, clinical depression,or grieving.

[0104] In another embodiment, antibodies which are specific for NHT maybe used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to a subject withan eating disorder including, but not limited to, those listed above.

[0105] In another embodiment, a vector expressing antisense of thepolynucleotide encoding NHT may be administered to a subject to to treator prevent an eating disorder including, but not limited to, thoselisted above.

[0106] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0107] Antagonists or inhibitors of NHT may be produced using methodswhich are generally known in the art. In particular, purified NHT may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind NHT.

[0108] Antibodies to NHT may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0109] For the production of antibodies to NHT, various hosts includinggoats, rabbits, rats, mice, humans, and others, may be immunized byinjection with NHT or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0110] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to NHT have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of NHT amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0111] Monoclonal antibodies to NHT may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0112] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceNHT-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0113] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0114] Antibody fragments which contain specific binding sites for NHTmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0115] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between NHT and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NHT epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0116] In another embodiment of the invention, the polynucleotidesencoding NHT, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding NHT may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding NHT. Thus, antisense molecules may be used to modulate NHTactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding NHT.

[0117] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encoding NHT.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0118] Genes encoding NHT can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes NHT. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0119] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding NHT, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0120] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NHT.

[0121] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0122] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding NHT. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0123] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0124] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0125] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0126] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of NHT,antibodies to NHT, mimetics, agonists, antagonists, or inhibitors ofNHT. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0127] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0128] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0129] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0130] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0131] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0132] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0133] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0134] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0135] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0136] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0137] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of NHT, such labeling wouldinclude amount, frequency, and method of administration.

[0138] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0139] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0140] A therapeutically effective dose refers to that amount of activeingredient, for example NHT or fragments thereof, antibodies of NHT,agonists, antagonists or inhibitors of NHT, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0141] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0142] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0143] Diagnostics

[0144] In another embodiment, antibodies which specifically bind NHT maybe used for the diagnosis of conditions or diseases characterized byexpression of NHT, or in assays to monitor patients being treated withNHT, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NHT includemethods which utilize the antibody and a label to detect NHT in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0145] A variety of protocols including ELISA, RIA, and FACS formeasuring NHT are known in the art and provide a basis for diagnosingaltered or abnormal levels of NHT expression. Normal or standard valuesfor NHT expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to NHT under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of NHTexpressed in control and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0146] In another embodiment of the invention, the polynucleotidesencoding NHT may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNHT may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of NHT, andto monitor regulation of NHT levels during therapeutic intervention.

[0147] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NHT or closely related molecules, may be used to identifynucleic acid sequences which encode NHT. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NHT, alleles, or related sequences.

[0148] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the NHT encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO: 2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring NHT.

[0149] Means for producing specific hybridization probes for DNAsencoding NHT include the cloning of nucleic acid sequences encoding NHTor NHT derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0150] Polynucleotide sequences encoding NHT may be used for thediagnosis or monitoring of appetite and eating disorders, especiallythose associated with amenorrhea; anorexia nervosa; bulimia nervosa;cachexia; loss of appetite associated with chemotherapy, clinicaldepression, or grieving; cardiovascular diseases such asarteriosclerosis, atheroschlerosis, and hypertension, autoimmune orgenetic disorders such as asthma, fatty liver or pancreas, hypoglycemia,hyperglycemia, and diabetes mellitus, gall bladder disease;hyperlipidaemia; Alstrom, Bardet-Biedl, Cushing's, Froehlich's, andPrader-Willi syndromes; sleep apnea; and adenocarcinomas, leukemias,lymphomas, melanomas, or sarcomas, particularly craniopharyngioma,hypothalmic, pituitary, and islet cell tumors, and adenocarcinomas ofthe brain, breast, gall bladder, liver, pancreas, and prostate.

[0151] The polynucleotide sequences encoding NHT may be used in Southernor northern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered NHTexpression. Such qualitative or quantitative methods are well known inthe art.

[0152] In a particular aspect, the nucleotide sequences encoding NHT maybe useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding NHT may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding NHT in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0153] In order to provide a basis for the diagnosis of diseaseassociated with expression of NHT, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes NHT, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0154] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0155] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0156] Additional diagnostic uses for oligonucleotides designed from thesequences encoding NHT may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′→3′) and another with antisense(3′←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0157] Methods which may also be used to quantitate the expression ofNHT include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0158] In another embodiment of the invention, the nucleic acidsequences which encode NHT may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0159] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding NHT on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0160] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0161] In another embodiment of the invention, NHT, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between NHTand the agent being tested, may be measured.

[0162] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to NHT large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NHT, or fragments thereof, and washed. Bound NHT is thendetected by methods well known in the art. Purified NHT can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0163] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding NHTspecifically compete with a test compound for binding NHT. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with NHT.

[0164] In additional embodiments, the nucleotide sequences which encodeNHT may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0165] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0166] I HNT2NOT01 cDNA Library Construction

[0167] The hNT2 cell line exhibits characteristics of a committedneuronal precursor cell which is still at an early stage of development.The cDNA library (HNT2NOT01; Cat. No. 937230) was prepared fromuntreated hNT2 cell line by Stratagene (La Jolla, Calif.).

[0168] cDNAs were primed using oligo d(T) and size fractionated toisolate fragments of 500 bp and larger. Synthetic adapteroligonucleotides were ligated onto the cDNA molecules enabling them tobe inserted into the UNI-ZAP vector system (Stratagene).

[0169] The quality of the cDNA library was screened using DNA probes,and then, the PBLUESCRIPT phagemid (Stratagene) was excised.Subsequently, the phagemid was transfected into E. coli host strainXL1-BLUE (Stratagene). Alternative unidirectional vectors include, butare not limited to, PCDNAI (Invitrogen, San Diego Calif.) and PSHLOX-1(Novagen, Madison Wis.).

[0170] II Isolation of cDNA Clones

[0171] The phagemids containing individual cDNA clones were isolatedwith the Miniprep Kit (Cat. No. 77468; Advanced Genetic TechnologiesCorp., Gaithersburg Md.). This kit has a 96-well format and providesenough reagents for 960 purifications. The recommended protocol wasemployed except for the following changes. First, the 96 wells were eachfilled with only 1 ml of sterile terrific broth with carbenicillin at 25mg/L and glycerol at 0.4%. After the wells were inoculated, the bacteriawere cultured for 24 hours and lysed with 60 μL of lysis buffer. Acentrifugation at 2900 rpm for 5 minutes was performed before thecontents of the block were added to the primary filter plate. Theoptional step of adding isopropanol to Tris buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

[0172] Alternatively, the in vivo excision process, in which the hostbacterial strain is co-infected with both the library phage and an f1helper phage, was used to harvest the phagemid. Polypeptides or enzymesderived from both the library-containing phage and the helper phagenicked the DNA, initiated new DNA synthesis from defined sequences onthe target DNA, and created a smaller, single stranded circular phagemidDNA molecule. The circular molecule included all DNA sequences of thePBLUESCRIPT phagemid and the cDNA insert. When the phagemid DNA wasreleased from the cells, it was purified, and used to reinfect freshhost cells (SOLR, Stratagene) which produced double-stranded DNA.Because the phagemid carries the gene for §-lactamase, the newlytransformed bacteria were selected on medium containing ampicillin.

[0173] Phagemid DNA may also be purified using the QIAWELL-8 plasmidpurification system (QIAGEN Inc., Chatsworth Calif.). This productprovides a convenient, rapid and reliable high-throughput method forlysing the bacterial cells and isolating highly purified phagemid DNA.The DNA is eluted from the purification resin and prepared for DNAsequencing and other analytical manipulations.

[0174] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0175] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc., Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0176] Peptide and protein sequence homologies were ascertained usingthe INHERIT-670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0177] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990)J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0178] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0179] IV Northern Analysis

[0180] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0181] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0182] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0183] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0184] The results of northern analysis are reported as a list oflibraries in which the transcript encoding NHT occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0185] V Extension of NHT Polynucleotide Sequences

[0186] The nucleic acid sequence of Incyte Clone 492199 is used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer is synthesized to initiate extensionin the antisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers are used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers are designed from the cDNA using OLIGO 4.06 primeranalysis software (National Biosciences), or another appropriateprogram, to be 22-30 nucleotides in length, to have a GC content of 50%or more, and to anneal to the target sequence at temperatures about68°-72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations is avoided.

[0187] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0188] By following the instructions for the XL-PCR kit (AppliedBiosystems) and thoroughly mixing the enzyme and reaction mix, highfidelity amplification is obtained. Beginning with 40 pmol of eachprimer and the recommended concentrations of all other components of thekit, PCR is performed using the Peltier thermal cycler (PTC200; M. J.Research, Watertown, Mass.) and the following parameters: Step 1 94° C.for 1 min (initial denaturation) Step 2 65° C. for 1 min Step 3 68° C.for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C.for 7 min Step 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C.for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C.(and holding)

[0189] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK DNA purification kit (QIAGEN). Afterrecovery of the DNA, Klenow enzyme is used to trim single-stranded,nucleotide overhangs creating blunt ends which facilitate religation andcloning.

[0190] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

[0191] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0192] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0193] In like manner, the nucleotide sequence of SEQ ID NO: 2 can beused to obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0194] VI Labeling and Use of Hybridization Probes

[0195] Hybridization probes derived from SEQ ID NO: 2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN).

[0196] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (NYTRAN PLUS membrane, Schleicher& Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at40° C. To remove nonspecific signals, blots are sequentially washed atroom temperature under increasingly stringent conditions up to 0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARautoradiography film (Kodak, Rochester, N.Y.) is exposed to the blots,or the blots are placed in a PHOSPHOIMAGER cassette (Molecular Dynamics,Sunnyvale, Calif.) for several hours, hybridization patterns arecompared visually.

[0197] VII Complementary Nucleic Acid Molecules

[0198] Antisense molecules or nucleic acid sequences complementary tothe NHT-encoding sequence, or any part thereof, are used to inhibit invivo or in vitro expression of naturally occurring NHT. Although use ofantisense oligonucleotides, comprising about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. An oligonucleotide based on the coding sequencesof NHT, as shown in FIGS. 1A and 1B, is used to inhibit expression ofnaturally occurring NHT. The complementary oligonucleotide is designedfrom the most unique 5′ sequence as shown in FIGS. 1A and 1B and usedeither to inhibit transcription by preventing promoter binding to theupstream nontranslated sequence or translation of an NHT-encodingtranscript by preventing the ribosome from binding. Using an appropriateportion of the signal and 5′ sequence of SEQ ID NO: 2, an effectiveantisense oligonucleotide includes any 15-20 nucleotides spanning theregion which translates into the signal or 5′ coding sequence of thepolypeptide as shown in FIGS. 1A and 1B.

[0199] VIII Expression of NHT

[0200] Expression of NHT is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, previously used for the generation of thecDNA library is used to express NHT in E. coli. Upstream of the cloningsite, this vector contains a promoter for β-galactosidase, followed bysequence containing the amino-terminal Met, and the subsequent sevenresidues of β-galactosidase. Immediately following these eight residuesis a bacteriophage promoter useful for transcription and a linkercontaining a number of unique restriction sites.

[0201] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of NHT into the bacterial growth media which can be useddirectly in the following assay for activity.

[0202] IX Demonstration of NHT Activity

[0203] The ability of NHT to stimulate appetite and eating can be testedin mice. The amount of food consumed per day, the average weight gain ina particular time period, the adipose deposition in females versus malesand the differences in weight gain between litter mates receiving or notreceiving the protein can be compared.

[0204] X Production of NHT Specific Antibodies

[0205] NHT that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO: 2 is analyzed using DNASTARsoftware (DNASTAR Inc.) to determine regions of high immunogenicity anda corresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0206] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431A peptide synthesizer(Applied Biosystems) using fmoc-chemistry, and coupled to keyhole limpethemocyanin (KLH, Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0207] XI Purification of Naturally Occurring NHT Using SpecificAntibodies

[0208] Naturally occurring or recombinant NHT is substantially purifiedby immunoaffinity chromatography using antibodies specific for NHT. Animmunoaffinity column is constructed by covalently coupling NHT antibodyto an activated chromatographic resin, such as CnBr-activated SEPHAROSE(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0209] Media containing NHT is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of NHT (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NHT binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and NHT is collected.

[0210] XII Identification of Molecules Which Interact with NHT

[0211] NHT or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133:529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled NHT, washed and any wells withlabeled NHT complex are assayed. Data obtained using differentconcentrations of NHT are used to calculate values for the number,affinity, and association of NHT with the candidate molecules.

[0212] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and to system of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 491 amino acids amino acid single linear peptide HNT2NOT01 Concensus1 Met Glu Ala Ser Arg Cys Arg Leu Ser Pro Ser Gly Asp Ser Val Phe 1 5 1015 His Glu Glu Met Met Lys Met Arg Gln Ala Lys Leu Asp Tyr Gln Arg 20 2530 Leu Leu Leu Glu Lys Arg Gln Arg Lys Lys Arg Leu Glu Pro Phe Met 35 4045 Val Gln Pro Asn Pro Glu Ala Arg Leu Arg Arg Ala Lys Pro Arg Ala 50 5560 Ser Asp Glu Gln Thr Pro Leu Val Asn Cys His Thr Pro His Ser Asn 65 7075 80 Val Ile Leu His Gly Ile Asp Gly Pro Ala Ala Val Leu Lys Pro Asp 8590 95 Glu Val His Ala Pro Ser Val Ser Ser Ser Val Val Glu Glu Asp Ala100 105 110 Glu Asn Thr Val Asp Thr Ala Ser Lys Pro Gly Leu Gln Glu ArgLeu 115 120 125 Gln Lys His Asp Ile Ser Glu Ser Val Asn Phe Asp Glu GluThr Asp 130 135 140 Gly Ile Ser Gln Ser Ala Cys Leu Glu Arg Pro Asn SerAla Ser Ser 145 150 155 160 Gln Asn Ser Thr Asp Thr Gly Thr Ser Gly SerAla Thr Ala Ala Gln 165 170 175 Pro Ala Asp Asn Leu Leu Gly Asp Ile AspAsp Leu Glu Asp Phe Val 180 185 190 Tyr Ser Pro Ala Pro Gln Gly Val ThrVal Arg Cys Arg Ile Ile Arg 195 200 205 Asp Lys Arg Gly Met Asp Arg GlyLeu Phe Pro Thr Tyr Tyr Met Tyr 210 215 220 Leu Glu Lys Glu Glu Asn GlnLys Ile Phe Leu Leu Ala Ala Arg Lys 225 230 235 240 Arg Lys Lys Ser LysThr Ala Asn Tyr Leu Ile Ser Ile Asp Pro Val 245 250 255 Asp Leu Ser ArgGlu Gly Glu Ser Tyr Val Gly Lys Leu Arg Ser Asn 260 265 270 Leu Met GlyThr Lys Phe Thr Val Tyr Asp Arg Gly Ile Cys Pro Met 275 280 285 Lys GlyArg Gly Leu Val Gly Ala Ala His Thr Arg Gln Glu Leu Ala 290 295 300 AlaIle Ser Tyr Glu Thr Asn Val Leu Gly Phe Lys Gly Pro Arg Lys 305 310 315320 Met Ser Val Ile Ile Pro Gly Met Thr Leu Asn His Lys Gln Ile Pro 325330 335 Tyr Gln Pro Gln Asn Asn His Asp Ser Leu Leu Ser Arg Trp Gln Asn340 345 350 Arg Thr Met Glu Asn Leu Val Glu Leu His Asn Lys Ala Pro ValTrp 355 360 365 Asn Ser Asp Thr Gln Ser Tyr Val Leu Asn Phe Arg Gly ArgVal Thr 370 375 380 Gln Ala Ser Val Lys Asn Phe Gln Ile Val His Lys AsnAsp Pro Asp 385 390 395 400 Tyr Ile Val Met Gln Phe Gly Arg Val Ala AspAsp Val Phe Thr Leu 405 410 415 Asp Tyr Asn Tyr Pro Leu Cys Ala Val GlnAla Phe Gly Ile Gly Leu 420 425 430 Ser Ser Phe Asp Lys Arg Ile Gln ThrLeu Arg Met Gln Glu Leu Cys 435 440 445 Glu Leu His Arg Gln His His SerAla Ala Ser Leu Val His Arg Thr 450 455 460 Ala Cys Gln Arg Trp Val GlyHis Pro Trp Arg Gln Leu Pro Gln Ser 465 470 475 480 Ser Leu Val Gly ProAsp Leu Xaa Leu His Met 485 490 1525 base pairs nucleic acid singlelinear cDNA HNT2NOT01 Concensus 2 GCACGAGGTG GGGGCTTTCC TCGGTGGCGGGCATGGAGGC TTCGCGCTGC CGGCTCAGT 60 CCAGCGGCGA CAGTGTCTTC CATGAAGAAATGATGAAGAT GCGACAGGCT AAGCTGGA 120 ATCAGAGGCT ACTACTTGAG AAGAGGCAAAGGAAAAAGCG CCTTGAGCCA TTTATGGT 180 AGCCCAATCC AGAAGCCAGG CTACGTCGGGCAAAGCCAAG GGCCAGTGAT GAGCAGAC 240 CCTTGGTGAA CTGTCATACT CCCCACAGCAATGTCATCTT ACATGGTATT GATGGTCC 300 CTGCTGTCCT GAAACCAGAC GAAGTTCATGCTCCATCAGT AAGCTCCTCT GTTGTGGA 360 AAGATGCTGA AAACACCGTG GATACTGCTTCCAAGCCAGG ACTTCAGGAG CGTCTCCA 420 AGCATGATAT CTCTGAAAGT GTGAACTTCGATGAGGAGAC TGATGGAATA TCCCAGTC 480 CATGTTTAGA AAGACCCAAT TCTGCATCAAGCCAGAATTC AACCGATACA GGCACTTC 540 GTTCTGCTAC TGCCGCCCAA CCAGCTGATAACCTCCTGGG AGACATAGAC GACCTGGA 600 ACTTTGTGTA TAGTCCTGCC CCTCAAGGTGTCACAGTAAG ATGTCGGATA ATCCGGGA 660 AAAGGGGAAT GGATCGGGGT CTCTTCCCCACCTACTATAT GTACTTGGAA AAAGAAGA 720 ATCAGAAGAT ATTTCTTCTT GCAGCTAGAAAGCGGAAAAA GAGCAAAACA GCCAACTA 780 TTATCTCCAT TGATCCAGTT GATTTATCTCGTGAAGGAGA AAGTTATGTC GGCAAGCT 840 GATCCAACCT CATGGGGACC AAGTTTACAGTTTATGACCG TGGCATCTGC CCCATGAA 900 GCCGGGGTTT GGTAGGAGCG GCCCACACCCGGCAGGAGCT GGCTGCCATC TCCTATGA 960 CAAACGTACT TGGATTTAAA GGTCCTAGGAAAATGTCTGT GATCATTCCT GGAATGA 1020 TGAATCATAA GCAGATCCCC TATCAGCCACAAAACAACCA TGACAGTTTG CTCTCAA 1080 GGCAGAACAG AACTATGGAA AATCTGGTTGAGCTGCACAA CAAGGCCCCC GTCTGGA 1140 GTGACACTCA GTCCTATGTC CTCAACTTCCGTGGCCGGGT CACTCAGGCG TCTGTGA 1200 ACTTCCAGAT AGTCCACAAA AATGACCCTGATTATATAGT CATGCAGTTT GGACGTG 1260 CAGATGACGT GTTCACACTG GATTACAACTACCCACTTTG TGCAGTACAG GCCTTTG 1320 TCGGTCTTTC TAGCTTTGAC AAACGTATCCAAACCTTGAG AATGCAGGAG CTCTGTG 1380 TCCACCGTCA GCACCATTCA GCTGCATCCCTTGTGCACAG GACTGCCTGC CAGCGTT 1440 TGGGACACCC GTGGCGGCAG CTCCCTCAGTCTTCCCTTGT CGGCCCTGAC CTNTNAC 1500 ATATGTAGNA GCCCGAGACC AAAAA 1525 505amino acids amino acid single linear peptide GenBank 1279766 3 Met ThrSer Lys Pro His Ser Asp Trp Ile Pro Tyr Ser Val Leu Asp 1 5 10 15 AspGlu Gly Ser Asn Leu Arg Gln Gln Lys Leu Asp Arg Gln Arg Ala 20 25 30 LeuLeu Glu Gln Lys Gln Lys Lys Lys Arg Gln Glu Pro Leu Met Val 35 40 45 GlnAla Asn Ala Asp Gly Arg Pro Arg Ser Arg Arg Ala Arg Gln Ser 50 55 60 GluGlu Gln Ala Pro Leu Val Glu Ser Tyr Leu Ser Ser Ser Gly Ser 65 70 75 80Thr Ser Tyr Gln Val Gln Glu Ala Asp Ser Ile Ala Ser Val Gln Leu 85 90 95Gly Ala Thr Arg Pro Pro Ala Pro Ala Ser Ala Lys Lys Ser Lys Gly 100 105110 Ala Ala Ala Ser Gly Gly Gln Gly Gly Ala Pro Arg Lys Glu Lys Lys 115120 125 Gly Lys His Lys Gly Thr Ser Gly Pro Ala Thr Leu Ala Glu Asp Lys130 135 140 Ser Glu Ala Gln Gly Pro Val Gln Ile Leu Thr Val Gly Gln SerAsp 145 150 155 160 His Asp Lys Asp Ala Gly Glu Thr Ala Ala Gly Gly GlyAla Gln Pro 165 170 175 Ser Gly Gln Asp Leu Arg Ala Thr Met Gln Arg LysGly Ile Ser Ser 180 185 190 Ser Met Ser Phe Asp Glu Asp Glu Asp Glu AspGlu Asn Ser Ser Ser 195 200 205 Ser Ser Gln Leu Asn Ser Asn Thr Arg ProSer Ser Ala Thr Ser Arg 210 215 220 Lys Ser Ile Arg Glu Ala Ala Ser AlaPro Ser Pro Ala Ala Pro Glu 225 230 235 240 Pro Pro Val Asp Ile Glu ValGln Asp Leu Glu Glu Phe Ala Leu Arg 245 250 255 Pro Ala Pro Gln Gly IleThr Ile Lys Cys Arg Ile Thr Arg Asp Lys 260 265 270 Lys Gly Met Asp ArgGly Met Tyr Pro Thr Tyr Phe Leu His Leu Asp 275 280 285 Arg Glu Asp GlyLys Lys Val Phe Leu Leu Ala Gly Arg Lys Arg Lys 290 295 300 Lys Ser LysThr Ser Asn Tyr Leu Ile Ser Val Asp Pro Thr Asp Leu 305 310 315 320 SerArg Gly Gly Asp Ser Tyr Ile Gly Lys Leu Arg Ser Asn Leu Met 325 330 335Gly Thr Lys Phe Thr Val Tyr Asp Asn Gly Val Asn Pro Gln Lys Ala 340 345350 Ser Ser Ser Thr Leu Glu Ser Gly Thr Leu Arg Gln Glu Leu Ala Ala 355360 365 Val Cys Tyr Glu Thr Asn Val Leu Gly Phe Lys Gly Pro Arg Lys Met370 375 380 Ser Val Ile Val Pro Gly Met Asn Met Val His Glu Arg Val CysIle 385 390 395 400 Arg Pro Arg Asn Glu His Glu Thr Leu Leu Ala Arg TrpGln Asn Lys 405 410 415 Asn Thr Glu Ser Ile Ile Glu Leu Gln Asn Lys ThrPro Val Trp Asn 420 425 430 Asp Asp Thr Gln Ser Tyr Val Leu Asn Phe HisGly Arg Val Thr Gln 435 440 445 Ala Ser Val Lys Asn Phe Gln Ile Ile HisGly Asn Asp Pro Asp Tyr 450 455 460 Ile Val Met Gln Phe Gly Arg Val AlaGlu Asp Val Phe Thr Met Asp 465 470 475 480 Tyr Asn Tyr Pro Leu Cys AlaLeu Gln Ala Phe Ala Ile Ala Leu Ser 485 490 495 Ser Phe Asp Ser Lys LeuAla Cys Glu 500 505 506 amino acids amino acid single linear proteinGenBank 1305497 4 Met Thr Ser Lys Pro His Ser Asp Trp Ile Pro Tyr SerVal Leu Asp 1 5 10 15 Asp Glu Gly Arg Asn Leu Arg Gln Gln Lys Leu AspArg Gln Arg Ala 20 25 30 Leu Leu Glu Gln Lys Gln Lys Lys Lys Arg Gln GluPro Leu Met Val 35 40 45 Gln Ala Asn Ala Asp Gly Arg Pro Arg Ser Arg ArgAla Arg Gln Ser 50 55 60 Glu Glu Gln Ala Pro Leu Val Glu Ser Tyr Leu SerSer Ser Gly Ser 65 70 75 80 Thr Ser Tyr Gln Val Gln Glu Ala Asp Ser LeuAla Ser Val Gln Leu 85 90 95 Gly Ala Thr Arg Pro Thr Ala Pro Ala Ser AlaLys Arg Thr Lys Ala 100 105 110 Ala Ala Thr Ala Gly Gly Gln Gly Gly AlaAla Arg Lys Glu Lys Lys 115 120 125 Gly Lys His Lys Gly Thr Ser Gly ProAla Ala Leu Ala Glu Asp Lys 130 135 140 Ser Glu Ala Gln Gly Pro Val GlnIle Leu Thr Val Gly Gln Ser Asp 145 150 155 160 His Ala Gln Asp Ala GlyGlu Thr Ala Ala Gly Gly Gly Glu Arg Pro 165 170 175 Ser Gly Gln Asp LeuArg Ala Thr Met Gln Arg Lys Gly Ile Ser Ser 180 185 190 Ser Met Ser PheAsp Glu Asp Glu Glu Asp Glu Glu Glu Asn Ser Ser 195 200 205 Ser Ser SerGln Leu Asn Ser Asn Thr Arg Pro Ser Ser Ala Thr Ser 210 215 220 Arg LysSer Val Arg Glu Ala Ala Ser Ala Pro Ser Pro Thr Ala Pro 225 230 235 240Glu Gln Pro Val Asp Val Glu Val Gln Asp Leu Glu Glu Phe Ala Leu 245 250255 Arg Pro Ala Pro Gln Gly Ile Thr Ile Lys Cys Arg Ile Thr Arg Asp 260265 270 Lys Lys Gly Met Asp Arg Gly Met Tyr Pro Thr Tyr Phe Leu His Leu275 280 285 Asp Arg Glu Asp Gly Lys Lys Val Phe Leu Leu Ala Gly Arg LysArg 290 295 300 Lys Lys Ser Lys Thr Ser Asn Tyr Leu Ile Ser Val Asp ProThr Asp 305 310 315 320 Leu Ser Arg Gly Gly Asp Ser Tyr Ile Gly Lys LeuArg Ser Asn Leu 325 330 335 Met Gly Thr Lys Phe Thr Val Tyr Asp Asn GlyVal Asn Pro Gln Lys 340 345 350 Ala Ser Ser Ser Thr Leu Glu Ser Gly ThrLeu Arg Gln Glu Leu Ala 355 360 365 Ala Val Cys Tyr Glu Thr Asn Val LeuGly Phe Lys Gly Pro Arg Lys 370 375 380 Met Ser Val Ile Val Pro Gly MetAsn Met Val His Glu Arg Val Ser 385 390 395 400 Ile Arg Pro Arg Asn GluHis Glu Thr Leu Leu Ala Arg Trp Gln Asn 405 410 415 Lys Asn Thr Glu SerIle Ile Glu Leu Gln Asn Lys Thr Pro Val Trp 420 425 430 Asn Asp Asp ThrGln Ser Tyr Val Leu Asn Phe His Gly Arg Val Thr 435 440 445 Gln Ala SerVal Lys Asn Phe Gln Ile Ile His Gly Asn Asp Pro Asp 450 455 460 Tyr IleVal Met Gln Phe Gly Arg Val Ala Glu Asp Val Phe Thr Met 465 470 475 480Asp Tyr Asn Tyr Pro Leu Cys Ala Leu Gln Ala Phe Ala Ile Ala Leu 485 490495 Ser Ser Phe Asp Ser Lys Leu Ala Cys Glu 500 505

What is claimed is:
 1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence of SEQ ID NO: 1, b) a naturally-occurring amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 1, c) a biologically-active fragment of the amino acid sequence of SEQ ID NO: 1, and d) an immunogenic fragment of the amino acid sequence of SEQ ID NO:
 1. 2. An isolated polypeptide of claim 1, having a sequence of SEQ ID NO:
 1. 3. An isolated polynucleotide encoding a polypeptide of claim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. An isolated polynucleotide of claim 4, having a sequence of SEQ ID NO:
 2. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 3. 7. A cell transformed with a recombinant polynucleotide of claim
 6. 8. A method for producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
 9. A method of claim 8, wherein the polypeptide has the sequence of SEQ ID NO:
 1. 10. An isolated antibody which specifically binds to a polypeptide of claim
 1. 11. An isolated polynucleotide comprising a sequence selected from the group consisting of: a) a polynucleotide sequence of SEQ ID NO: 2, b) a naturally-occurring polynucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 2, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b) and e) a ribonucleotide equivalent of a)-d).
 12. An isolated polynucleotide comprising at least 60 contiguous nucleic acids of claim
 11. 13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.
 15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 16. A composition comprising a polypeptide of claim 1 and an acceptable excipient.
 17. A composition of claim 16, wherein the polypeptide has the sequence of SEQ ID NO:
 1. 18. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
 19. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
 20. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 11, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 21. A method for assessing toxicity of a test compound, said method comprising: a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 11 or fragment thereof; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 22. An isolated polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:
 1. 23. An isolated polynucleotide of claim 11, comprising the amino acid sequence of SEQ ID NO:
 2. 24. A diagnostic test for a condition or disease associated with the expression of NHT in a biological sample comprising the steps of: a) combining the biological sample with an antibody of claim 10, under conditions suitable for the antibody to bind the polypeptide and form an antibody: polypeptide complex; and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
 25. The antibody of claim 10, wherein the antibody is: (a) a chimeric antibody; (b) a single chain antibody; (c) a Fab fragment; (d) a F(ab′)₂ fragment; or (e) a humanized antibody.
 26. A composition comprising an antibody of claim 10 and an acceptable excipient.
 27. A method of diagnosing a condition or disease associated with the expression of NHT in a subject, comprising administering to said subject an effective amount of the composition of claim
 26. 28. A composition of claim 26, wherein the antibody is labeled.
 29. A method of diagnosing a condition or disease associated with the expression of NHT in a subject, comprising administering to said subject an effective amount of the composition of claim
 28. 30. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 10 comprising: a) immunizing an animal with a polypeptide of SEQ ID NO: 1 or an immunogenic fragment thereof under conditions to elicit an antibody response; b) isolating antibodies from said animal; and c) screening the isolated antibodies with the polypeptide thereby identifying a polyclonal antibody which binds specifically to a polypeptide of SEQ ID NO:
 1. 31. An antibody produced by a method of claim
 30. 32. A composition comprising the antibody of claim 31 and a suitable carrier.
 33. A method of making a monoclonal antibody with the specificity of the antibody of claim 10 comprising: a) immunizing an animal with a polypeptide of SEQ ID NO: 1 or an immunogenic fragment thereof under conditions to elicit an antibody response; b) isolating antibody producing cells from the animal; c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells; d) culturing the hybridoma cells; and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide of SEQ ID NO:
 1. 34. A monoclonal antibody produced by a method of claim
 33. 35. A composition comprising the antibody of claim 34 and a suitable carrier.
 36. The antibody of claim 10, wherein the antibody is produced by screening a Fab expression library.
 37. The antibody of claim 10, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 38. A method for detecting a polypeptide of SEQ ID NO: 1 in a sample comprising the steps of: a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide of SEQ ID NO: 1 in the sample.
 39. A method of purifying a polypeptide of SEQ ID NO: 1 from a sample, the method comprising: a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and b) separating the antibody from the sample and obtaining purified polypeptide of SEQ ID NO:
 1. 